P-glycoprotein 170 (P-gp 170), a member of the ABC (ATP Binding Cassette) family, acts as an ATP-dependent drug efflux pump, preventing intracellular accumulation of miscellaneous drugs.1,2 Overexpression of this protein is one of the mechanisms of multidrug resistance (MDR) of cancer cells. This protein is expressed in a cell- and tissue-specific manner, with high levels detectable in the kidney, liver, blood-brain barrier and lining of the intestine.3 Studies using mdrl knockout mice and P-gp 170 tissue distribution in humans suggested several physiological roles of P-gp 170, including protection against toxic xenobiotics by blocking absorption by intestine; excretion of chemicals into bile duct or kidney tubule; prevention of chemicals taken into the brain through the blood-brain barrier; and efflux of steroid hormones and cholesterol from fesces.2,4,5 Developing drugs to inhibit P-gp 170 activity is an important area of drug discovery. Such drugs could have use in facilitating the oral absorption of drugs through intestine, or the uptake of chemicals that are substrates of P-gp, into the brain. In addition, these compounds could also potentate the action of antitumor drugs, which are substrates of P-gp 170 in cancer cells that overexpress the P-gp 170 protein.
A large number of compounds with major structural differences have been found to act as inhibitors or substrates of P-gp 170: these include verapamil (VRM), a calcium channel antagonist; trifluoperazine, a calmodulin inhibitor; cyclosporin A (CSA), an immunosuppressant and progesterone, a steroid hormone. Verapamil has been examined clinically in combination with cancer chemotherapy.6,7 However, the results were rather unsatisfactory due to high plasma drug levels, required to effectively reverse the MDR phenotype of cancer cells, which could cause cardiac toxicity. Compounds with higher potency against, and selectivity for, P-gp 170 are needed. A second generation of MDR reversal agents has emerged, and is based on the chemical modification of the first generation of inhibitors. Among these, dexniguldipine8 and dexverapamil9 were found to be more selective against P-gp 170, but they did not display improved potency. The acridonecarboxamide derivative GF120918 (GG918)10 and the cyclosporin A analog PSC83311 both displayed an activity that was 10-30 times more potent than the first generation of modulators, such as verapamil, tamoxifen and cyclosporin A.12 A number of these compounds are currently under clinical evaluation.
Flavonoids are an important class of natural products found in plants. With its polyphenolic structure, this class of compounds has multiple actions, including interaction with estrogen receptor, free-radical scavenger, protein kinase inhibitor, NF-B inhibitor, P-gp 170 inhibitor, among others.13 The biological activity found in herbal preparations is often attributed to its flavonoids. For example, the co-administration of grapefruit juice with various drugs has led to an increase in the plasma concentration of the drugs, which was attributed to the bioflavonoids found in the grapefruit juice.14,15 
Flavonoids have also been shown to act on multiple targets with different specificity. For example, the flavonoid that binds to estrogen receptor requires hydroxyl groups at positions 2 and 3 of the B-ring, a double bond at positions 2-3 of the C-ring, and the absence of any hydrophobic prenylated substituent.16 This is markedly different than the flavonoids that inhibit various ATPases or protein kinases. Recognition of the ATP binding pocket of these proteins requires the presence of three hydroxyl groups at positions 5 and 7 on the A-ring and position 3 of the C-ring, which favors some flavonols.17 Moreover, some protein kinases exhibit different structural requirements for binding: an isoflavone structure has been demonstrated to inhibit tyrosine kinase activity,18 and a flavone substituted at position 8 of A-ring with 4-(3-hydroxy-1-methylpiperidinyl) group has demonstrated activity against CDK2.19 The inhibition of P-gp 170 by flavonoids has also been investigated and two binding modes have been postulated. The studies were performed using a truncated form of P-gp 170 in a membrane preparation from P-gp 170 overexpressing cells. The structural requirements for flavonoid activity have recently been summarized20, and it appears that different classes of flavonoids have different structural requirements for inhibitory activity against P-gp 170.
In our previous study, we found that Radix scutellariae (Scute), a well-known Chinese herb, has inhibitory activity against P-gp 170. The active component of the Scute herb was found to be the natural product baicalein. However, the 7-glucuronosyl form of baicalein, which is the most abundant component of Scute, did not show anti-P-gp 170 activity. Other abundant Scute components, such as wogonoside and wogonin, were also found to lack inhibitory activity against P-gp 170 efflux action. Since baicalein (a 5,6,7-trihydroxyflavone) and wogonin (a 5,7-dihydroxy-8-methoxyflavone) have a very similar structure, this raised the possibility of an interesting structure-activity relationship of the flavone natural products for P-gp 170 inhibition.
In this study, we synthesized and evaluated a series of baicalein analogs, focusing on the substitution pattern at positions 5, 6, 7 and 8 of the A-ring. The results of this substitution show that alkoxyl groups on the A-ring of the flavone greatly increase the anti-P-gp 170 activity and alter their selectivity for the efflux pump.